Process and culture media for producing new penicillins



"requirements of surprising in view of edema Aug. 1c, 1949 UNITED STATESPATENT OFFICE PROCESS AND PRODUCING N Otto K. Belu'ens, Joseph W.

Jones, and Quentin F. Soper, assignors to Eli Lilly and Company,

CULTURE MEDIA Fon EW PENICIILINS Corse, Reuben G. Indianapolis, Ind.,

Indian apolis, IndL, a corporation of Indiana No Drawing. ApplicationMarch a, 1940,

' Serial No. 653,137

Q penicillin-producing mold may be induced to produce a novelpenicillin, by incorporating in the nutrient medium wherein the mold isgrown, a selected organic compound, called herein a precursor compound.Such selected precursor compound, although foreign to the normalmetabolic the mold, may be metabolized and incorporated in substantialpart in the molecule of a novel penicillin. This result is especiallythe recognized specificity of the enzyme systems whereby lower organismsmaintain growth and development. It is further surprising that use or aselected precursor compound may lead to the production or a novel to thesubstantial exclusion or the known and normally-produced penicillins.

Thus, by this invention there are provided not only methods of producingnovel penicillins with- ,put substantial concomitant production of knownand normally-produced penicillins, but also there are provided methodsof producing novel penicillins which incorporate substantial portions ofselected procursor compo In accordance with one aspect of this inventiothere are provided novel penicillins. In accordance with another aspect,there are provided methods of obtaining novel penicillins by afermentative process, which comprises growing, a penicillin-producingmold in -a nutrient mediumin the presence of a selected precursorcompound which the mold may metabolize and incorporate it in substantialpart in the molecule of a novel penicillin.

The present invention in its composition aspect contemplates novelproducts or Iermentative processes which comprise growing apenicillinproducing mold in association with a culture medium containingnutrient material and a selected precursor compound, said product as produced consisting essentially or a penicillin represented by the formulawherein R represents an aliphatic radical other than the butene-l-yl,butene-2-yl, n-butyl and n-hexyl radicals; a carbocyclic-includingradical other than the phenyl and p-hydroxyphenyl radicals; or aheterocyclic-including radical. Thus, the aliphatic radicals which R mayrepresent include straight-chain, branched-chain, saturated, andunsaturated radicals, of which are the A -nonenyl, ally] and tertiarybutyi radicals. Additional aliphatic radicals which R may represent arethose which contain as a member of the chain an interrupting group suchas oxygen, sulfur, nitrogen and the like, for example the allyloxy andethylmercapto radicals. The carbocyclic-including radicals which It mayrepresent include fully saturated and partially or completelyunsaturated carbocyclic nuclei, illustrative examples of which are the5,6,'I,8-tetrahydro-2-naphthyl, cyclopentyl, and cyclohexenyi radicals.Additional carbocyclicincluding radicals which It may represent arethose wherein the carbocyclic nucleus is attached to the -C1oHiaN2O4Smoiety by an aliphatic group or by some atom or group not containingcarbon. Illustrative examples of such radicals are the benzylmercapto,phenoxy, styryl and 4x,- dimethylbenzyl radicals. Theheterocyclic-including radicals which B may represent includes thethienyl radical and heterocyclic-including radicals wherein theheterocyclic nucleus is attached to the -Cl0Hl3N204S moiety by analiphatic group or by some atom or group not containing carbon. Anillustrative example is the a-(2-thienyllethyl radical.

Further specific examples of aliphatic, carbocyclic-including andheterocyclic-including radicals falling within the ambit of thisinvention are the vinyl, ethoxy, cyclopentenyl, cyclohexyl, 2,4-dichlorophe'nyl, 3,4 dichlorophenyl, m bromophenyl, o-bromophenyl,p-brornophenyl, m-chlorophenyl, o-chlorophenyl, p-chlorophenyl,mfluorophenyl, o-fluorophenyl, p-fluorophenyl, piodophenyl,p-nitrophenyl, p-chloroplienoxy, m-

illustrative examples I nitrophenyl, o-nitrophenyl,3-chloro-4-bromophenyl, m triiluoromethylphenyl, mtrifiuoromethylphenoxy. p-tolyl. omethylphenyl, mmethylphenyl,phenylmercapto, p-methylmercaptophenyl, phenylseleno, o-methoxyphenyl,mmethoxyphenyl, o-methylphenoxy. p-methoxyphenoxy, p-cyanophenyl,styryl, -3,4-dimethylphenyl, p-phenylbutyryl, p-carbethoxyhydroxyphenyl,p-naphthyl, p-naphthoxy, 1-bromo-2- naphthyl, 6-bromo-2-naphthyl,2-chloro-3-naphthyl, 6-iluoro-2-naphthyl, 1-nitro-2-naphthyl,naphthylmercapto, 6 -methoxy-2-naphthyl,' pphenoxyphenyl,p-isopropylphenyl and p-benzyloxyphenyl radicals.

For convenience, the new penicillins are named by reference to theparticular R radical contained therein. Thus, for example, a penicillinwherein the R of the next preceding formula. is the 2-thienyl radical isnamed 2-thienyl-penicillin.

In accordance with the method aspect of this invention, methods areprovided whereby novel penicillins are produced by growing apenicillinproducing mold in a culture medium containing nutrientmaterial and a selected precursor. compound which the mold maymetabolize and incorporate in substantial part in the molecule oi anovel penicillin. Precursor compounds useful for the purposes of thisinvention comprise monosubstituted acetic acids represened by theformula RCH2COOH wherein R represents an aliphatic radical other thanthe butene-l-yl, butene-Z-yl, n-butyl and nhexyl radicals; acarbocyclic-includlng radical other than the phenyl and p-hydroxypheny1radicals; or a heterocyclic-including radical. In place of themonosubstituted acetic acids represented by the formula hereinabovethere may be used equivalents of such acetic acids, said equivalentscomprising those compounds readily converted by the mold to themonosubstituted acetic acids. Such equivalents include simplederivatives of the acids such as their salts, esters. amides andanhyrides, as well as iii-substituted, saturated.

straight chain alcohols, amines, aldehydes and acids containing an evennumber of carbon atoms. and the simple derivatives thereof, all of whichthe mold may convert to the monosubstituted acetic acids. Examples ofequivalents include sodium fi-naphthylacetate, N- (Z-thienyl)acetylvaline, fl p-tolylethanol, N-(Z-hydroxyethyl) -'y (p bromophenyl)-butyramide, fl-chloropropionaldehyde diethylacetal,p-methoxy-phenethylamine hydrochloride and ethyl vinylacetate.

Broadly speaking, a method of producing a novel penicillin in accordancewith this invention is as follows: There is provided a nutrient mediumsuitable for the growth of a penicillin-producing mold. To the nutrientmedium is added in effective amount a precursor compound comprising amonosubstituted acetic acid or its equivalent. The culture mediumcomposition comprising nutrient material and precursor compound isinoculated with a penicillin-poducing mold and the mold is grown underpenicillin-producing conditions, during which growth a new penicillin isproduced by the mold as a metabolic product. .After mold growth the moldmycelium is separated from the culture medium, and from the latter thenovel penicillin is separated.

The isolation of the new penicillin may be effected by methods known tothe art, such as adsorption and extraction to obtain a productsufficiently pure for practical purposes. If a purer product is desired,the new penicillin may be subabsorption, X-ray diflraction andantibacterial fi The nutrient material used in the composition whereinthe mold is grown may comprise ingredients such as water, sugars,inorganic salts and desirably one or more indeterminate compositionssuch as corn steep amino acids and bran. Numerous suitable nutrientmedia comprising materials of the type mentioned are known to the art.

During the growth of the mold the culture medium comprising nutrientmaterial and precursor compound is maintained at a suitable temperature,for example, in the range of 20-30 C. The range of temperature which hasbeen found: to be particularly suitable is from 24-26 C. The period oftime during which the mold isgrown will depend upon the objectivedesired. Thus the mold may be grown only during the period of itsmaximum rate of growth before mold growth is interrupted preliminary toisolating the new pen-. icillin. Such a period generally is from 2 to 3days; On the other hand, the mold may be grown fora longer period oftime to obtain the maximum yield of new penicillin, In such a case; moldgrowth is usually continued for about five days.

The mold may be grown under various condi tions. For example, the moldmay be grown-without agitation of the culture medium, in which case themold grows on the surface of the medium. Alternatively the culturemediummay be agitated by shaking or stirring during the growth of the,mold in which case the mold is dispersed through-;. out the culturemedium and grows while so dis-- persed.

The molds suitable for the purposes of invention are mold organisms ofthe type capable of producing penicillins. Such organisms include.

molds of the Penicillium notatum-chrusogenum. group as well as certainmolds of the Aspergillus. group. It is to be understood that not allmoldstrain G147 oi the Aspergillus flavus group.

used, and that this optimum The concentration of the precursorcompounds. employed in the culture medium may. vary over a substantialrange. may be present in the culture medium in concen-- trations to theorder of about 1 percent, but it is usually desirable that smallerconcentrations be employed since there is no particular-advantage to begained in employing concentrations in substantial excess )f thosenecessary to produce the optimum effect. It appears at present that theoptimum concentration ofthe monosubstituted acetic acids and derivativesthereof lies in the range of about 0.01 to about 0.05 percent on aweight volume basis when mold strain X1612 is range upwardly when moldstrain Q-176' is used;

The precursor compound may be associatedwith the mold and culture mediumat any suitable time. Thus the materials of the nutrient medium Theprecursor compoundsi concentration may may be inoculated with the moldand the precursor compound to be employed may be incorporated eitherbefore or after inoculation of the culture medium with the mold.

The molecular structures of the precursor compounds have an importanteffect in determining what portions of the precursor molecules will beincorporated in the new penicillin. Thus for example,p-bromophenylacetic acid, p-p-bromophenethanol, andY-(p-bromophenyD-butyric acid when incorporated in culture media.wherein the mold is grown, will each lead to the production of the samenew penicillin, namely, p-bromophenyl-penicillin. n the other hand, ithas been observed that certain molecular structures not only fail toproduce a new penicillin but have no apparent effect upon the metabolismof the mold when employed in the preferred concentra tion-s,Illustratively, B-(p-bromophenyD-propionic acid represents a compoundwhich in association with nutrient material fails to stimulate theproduction of a novel penicillin.

The following explanation is offered as to what occurs in the practiceof the method of producing new penicillins as herein disclosed, it beinunderstood that such explanation is not to be construed as in any wayaffecting the scope of the invention. It appears that the resultobtained can be attributed in part at least to an oxidation effected bythe mold, and in particular cases, to a degradation which is analogousto fl-oxidation if indeed it is not actually p-oxidation. Thus, it isbelieved that fi-p-bromophenethanol is enzymatically oxidized by themold to p-bromophenylacetic acid, the latter being the operativeprecursor compound. It is believed that 'y-(p-bromophenyl) -butyric acidundergoes a degradation of the aliphatic chain portion of its moleculewhich degradation is akin to p-oxidation, whereby it is converted top-bromophenylacetic acid which, as noted above, is utilized by the moldand incorporated in the new penicillin. On the other hand, degradationof p-(p-bromophenyD-proplonic acid by a process of B-degradation willnot result in the formation of a substituted acetic acid, but insteadwill form a substituted benzoic acid, namely, p-bromobenzoic acid.According to this concept, it is only monosubstituted acetic acids whichcan be utilized by the mold, and the degradation product ofp-bromophenylpropionic acid, not being a monosubstituted acetic acid butrather a benzoic acid, can have no effect in producing a new penicillin.Further substantiating this view is the result obtained with fl-phenyl-Bfi-dimethylpropionic acid whose use as a precursor compound will leadto the formation of a,a-dimethylbenzylpenicillin. According to thepresent concept, the presence of both th methyl and phenyl groups on the,B-carbon atom renders the molecule incapable of a fl-degradation, andaccordingly the compound represents an acetic acid which ismonosubstituted with an cad-dimethylbenzyl radical, and is utilized assuch. The presence of other groups such as unsaturated groups, orinterrupting atoms such as sulfur or oxygen, likewise may prevent thedegradation of a carbon chain. Thus for example, p-tolylmercaptoaceticacid which is analogous to p-(pmethylphenyl) propionic acid except thatin the aliphatic carbon chain a CH2-- group is replaced by an S group isnot degraded to the benzoic acid, butis utilized as such, and leads tothe formation. of p-tolylmeroapto-penicillin.

The present application is directed to novel culture media and theproduction of new penicillins by a method which comprises growing apenicillin-producing mold in a culture medium containing nutrientmaterial and a relatively small amount of a precursor compoundrepresented by the formula mine and aldehyde.

Subject matter disclosed but not claimed herein, is disclosed andclaimed in copending applications Serial Nos. 653,136, 653,138, 773,488,773,- 489 and 773,490. Applications Serial Nos. 773,488 through 773,490have been abandoned.

The following specific examples further illustrate the practice of thisinvention.

Example 1 The sodium salt of p-methoxyphenyl-penicillin represented bythe formula:

cmoQ-wmmmsm may be prepared in the following manner:

A culture medium ma be prepared in the following proportions:

Lactose a 125 Corn steep solids g Calcium carbonate g 10N-(2-hydroxyethyl) -p methoxyphenylacetamide g 0.84 Water cc 5,000

The culture medium is distributed in 200 cc. portions in 1 literErlenmeyer flasks, sterilized, inoculated with a spore suspension ofPenicillium mold, strain N. R. R. L. 1976, and stoppered with cottonplugs. The flasks are maintained at a temperature of about 23-26 C. andshaken constantly for five days. The flask contents are then filtered toremove the mold mycelium, the filtrate cooled to about 0 C., acidifiedto about pH 2.2 with orthophosphoric acid and shaken with an equalvolume of amyl acetate. The amyl acetate layer is separated andextracted with three successive 100 cc. portions of cold water to whichcold N/10 sodium bicarbonate solution is added during the course of eachextraction until a pH of about 7.0 to 7.3 is attained in the aqueousphase. The aqueous extracts are combined. cooled to about 0 C.,acidified to about pH 2.2 with orthophosphoric acid and extractedsuccessively with three 100 cc. portions of ether. The ether extractsare combined, and are passed through a chromatographic type silicaadsorption column about 30 mm. in diameter and 250 mm. long andcontaining a pH 6.2 phosphate buffer. The silica column is developed bypercolation with six 100 cc. portions of ether containing successivelyincreasing amounts of methanol in the order of 1, 1 /2, 2, 2 /2, and 3percent.

The developed silica columnv is divided into about 12 equal sections andeach section is eluted with three 30 cc. portions of M/15 phosphatebuffer of pH 7.0. The eluates are assayed bacteriologically to determinetheir penicillin content. 79 percent of the total antibiotic activitypossessed by all the eluates originates in a single band in .tone.

the silica coIumn and results from the presence ofp-methoxyphenyl-penicillin. This bandoccupies a position similar to thatin which penicillin G is found in comparable columns. Those eluatesrepresenting the sections of the silica column comprising this major,uniform band, are combined, cooled to about 0., acidified to about pH2.2 and extracted with three 50 cc. portions of chloroform. The combinedchloroform extracts are passed through a silica adsorption columncontaining a pH 6.2 phosphate buffer. This silica column is developed bypercolation with three 150 cc. portions of chloroform containingsuccessively increasing amounts of methanol in the order of 1, 2 and 3percent. The developed silica column is then divided into 12 equalsections and each section is eluted with three 30 cc. portions of M/15phosphate builfer of pH 7.0. Bacteriological assay shows that 92 percentof the total antibiotic activity originates in a single band in thesilica column. The eluates obtained by extraction of the silica columnsections which comprise this band are combined, cooled to about 0 C.,acidified to about pH 2.2 and extracted with three 100 cc, portions ofether. The ether extracts are combined and extracted with about '75 cc.of a cold, dilute aqueous solution of sodium hydroxide to which N/10sodium hydroxide is added during the course of the extraction so that afinal pH of about 7.0 is attained in the aqueous phase. From the aqueoussolution, the sodium salt of p-methoxyphenyl-penicillin may be separatedby any suitable means, for example, by freezing and evaporation in vacuofrom the frozen state.

The dry amorphous sodium salt of p-methoxyphenyl-penicillin istriturated with 1 cc. of acetone in which it almost completely dissolvesbut from which upon standing, it precipitates in crystalline form. Themixture is centrifuged and the sodium salt is washed with 5 cc. ofabsolute ace- The sodium salt is obtained in purified form by solutionin 1 cc. of 90 percent aqueous acetone and reprecipitation by theaddition of 4 cc. of absolute acetone.

The sodium salt of p-methoxyphenyl-penicillin prepared according to theabove procedure assayed about 1510 Oxford units per mg. when testedagainst Staph. aureus, strain 2092. A differential assay carried out onStaph. a-ureus strain 209P and B. subtilis strain N. R. R. L. B1558 gavea value of about 0.82. The optical rotation was found to be a value of7.9 as compared with a calculated value of 8.0.

Example 2 The sodium salt of p-methoxyphenyl-penicillln may also beprepared in the following manner:

A culture medium may be prepared in the following proportions:

Lactose Corn steep solids ...g 150 Calcium carbonate g 25p-Methoxyphenylacetic acid -g 0.66 Water cc 5,000

The culture medium is distributed in 200 cc.

portions in 1 liter Erlenmeyer flasks, sterilized,

inoculated with a spore suspension of Penicillium mold, strain X1612,and stoppered with cotton plugs. The flasks are maintained at a tempera-5 ture of about 23-26 C. and shaken constantly for five days. The flaskcontents are then filtered to remove the mold mycelium. Thep-methoxyphenyl-penicillin present in the filtrate may .be isolated andpurified according to the procedure described in Example 1.

The sodium salt of p-methoxyphenyl-peniclllin thus prepared possessesthe same characteristics and is identical with thep-methoxyphenyl-penicillin sodium salt prepared according to theprocedure given in Example 1.

Example 3 The sodium salt of p-methoxyphenyl-peniclllin may also beprepared by growing Penlcilllum mold, strain X1612, in arculture mediumof the following composition:

Lactose g 125 Corn steep solids g 150 Calcium carbonate g 25 Ethylp-methoxyphenyl acetate g 0.77 Water cc 5,000

The mold is grown in the culture medium and thep-methoxyphenyl-penicillin is isolated and purified according to theprocedure described in Example 1. q j

The sodium salt of p-methoxyphenyl peniciilin thus obtained is identicalwith the p-methoaqiphenyl-penicillin sodium salt obtained by theprocedure described in Example 1.

Eaample 4 The sodium salt of p-methoxyphenyl-penicillin may also beprepared in the following manner:

A culture medium is prepared as shown in Example 3 except that in placeof the 0.77 g. of ethyl p-methoxyphenylacetate there is employed 2.5 g.of p-methoxyphenylethylamin'e; The culture medium is distributed in 200cc. portions in 1 liter flasks, sterilized, and inoculated with a sporesuspension of Penicillium mold, strain X1612. The growth of the mold andisolation and purification of the p-methoxyphenylpenicillin are carriedout according to the procedure described in Example 1.

The sodium salt of p-methoxyphenyl-penicillin thus obtained is identicalwith that obtained by the procedure described in Example 1.

Example 5 The sodium salt of a-thienyl-penicillin represented by theformula HCCH may be prepared in the following manner:

A culture medium ma be prepared in the following proportions:

Lactose g 125 com steep solids g 100 Calcium carbonate ...g 10

N-(2 hydroxyethyl) a thienylaceta ide I 0.72 Water or 5,000 The culturemedium is distributed in 200 cc.

portions in 1 liter Erlenmeyer flasks. sterilized, inoculated with aspore suspension of Penicillium mold, strain N. R. R. L. 1976, andstoppered with cotton plugs. The flasks are maintained at a temperatureof about 23-26 C. and shaken constantly for live days. The flaskcontents are then filtered to remove the mold mycelium, the filtratecooled to about C., acidified to about pH 2.2 with orthophosphoric acidand shaken with about one half its volume of amyl acetate. The amylacetate layer is separated and extracted with three 100cc. portions ofcold water to which cold N/ sodium bicarbonate solution is added duringthe course of each extraction until a pH of about 7.1 to 7.3 is attainedin the a ueous phase. The aoueous extracts are combined,.cooled to about0 C., acidified to about pH 2.2 with orthophosphoric acid and extractedwith three 100 cc. portions of ether. The ether extracts are combined,dried over magnesium sulfate and are then passed through achromatographic type silica adsorption column about 30 mm. in diameterand 300 mm. long, and containing a pH 6.2 phosphate buffer. The silicacolumn is developed by percolation with six 100 cc. portions of ethercontaining successively increasing amounts of methanol in the order ofA2, 1, 1 /2, 2, 2 and 3 percent.

The developed silica column is divided into about 12 equal sections andeach section is eluted with three 30 cc. portions of M/ 15 phosphatebuffer of pH 7.0. The eluates are assayed bacteriologically to determinetheir penicillin content. 91 percent of the total antibiotic activitypossessed by all the eluates originates in a single band in the silicacolumn and results from the presence of a-thienyl-penicillin. This bandoccupies a po-.

sition similar to that in which penicillin G is found in comparablecolumns. The eluates obtained from the silica gel sections which make upthis uniform band are combined, cooled to about 0 C., acidified to aboutpH 2.2 with orthophosphoric acid and extracted with three 50 cc.portions of chloroform. The combined chloroform extracts are then passedthrough a silica adsorption column containing a pH 6.2 phosphate buffer.This silica gel column is developed by percolation with three 100 cc.portions of chloroform containing successively increasing amounts ofmethanol in the order of 1, 2 and 3 percent. The developed silica columnis then divided into 12 equal sections and each section is eluted withthree 30 cc. portions of M/ 15 phosphate buffer of pH 7.0.Bacteriological assay of the eluates shows 95 percent of the totalantibiotic activity concentrated in a single band in the silica column.The eluates obtained by extraction of the silica column sections whichcomprise this band are combined, cooled to about 0 0., acidified toabout pH 2.2 and extracted with three 100 cc. portions of ether. Theether extracts are combined and extracted with about 75 cc. of a colddilute aqueous solution of sodium hydroxide to which N/ 10 sodiumhydroxide solution is added during the course of the extraction so thata final pH of about 7.0 is obtained in the aqueous phase. From thisaqueous solution the sodium salt of a-thienyl-penicillin may be isolatedby any suitable means, for example, by freezing and evaporation in vacuofrom the frozen state.

The resulting dry sodium salt of the a-thienylpenicillin is washedseveral times with anhydrous acetone. The sodium salt is thencrystallized by dissolving it in 2 cc. of 90 percent acetone andreprecipitating it with 2 cc. of anhydrous acetone. 213 mg. of thesodium salt of m-thiBIiYl-PBDiOiHID is obtained.

The sodium salt of a-thienyl-penicillin prepared according to the aboveprocedure assayed about 1685 Oxford units per mg. when tested againstStaph. aureus strain 209P. A differential assay carried out on Staph.aureus, strain 2091', and B subtilis, strain N. R. R. L. 3-558, gave avalue of about 1.13. The optical rotation was found to be Example 6 Thesodium salt of p-chlorophenyl-penicillin represented by the formulaCIQCmHuNiOfiNa may be prepared in the following manner:

A culture medium may be prepared in the following proportions:

Lactose g 125 Corn steep solids g 100 Calcium carbonate g 10N-p-chlorophenylacetylvaline g 1.1 Water cc 5,000

The culture medium is distributed m 200 cc.

portions in 1 liter Erlenmeyer flasks, sterilized,

inoculated with a spore suspension of Penicillium mold, strain N. R. R.L. 1976, and stoppered with cotton plugs. The flasks are maintained at atemperature of about 23-26 C. and shaken constantly for five days. Theflask contents are then filtered to remove the mold mycelium, thefiltrate cooled to about 0 0., acidified to about pH 2.2

with orthophosphoric acid and shaken with an equal volume of amylacetate. The amyl acetate layer is separated and extracted with three100 cc. portions of cold water to which cold N/ 10 sodium bicarbonatesolution is added during the course of each extraction until a pH ofabout 7.1 to 7.3 is attained in the aqueous phase. The aqueous extractsare combined, cooled to about 0 C., acidifled to about pH 2.2 withorthophos'phoric acid and extracted with three 100 cc. portions ofether. The ether extracts are combined, and are passed through achromatographic type silica adsorption column about 30 mm. in diameterand 300 mm. long, and containing a pH 6.2 phosphate buffer. The silicacolumn is developed by percolation with six 100 cc. portions of ethercontaining successively increasing amount of methanol in the order of 1,1 2. 2%, and 3 percent.

The developed silica column is divided into about 12 equal sections andeach section is eluted with three 30 cc. portions of M/ 15 phosphatebuffer of pH 7.0. The eluates are assayed bacteriologically to determinetheir penicillin content. About 92 percent of the total antibioticactivity possessed by all the eluates originates in a single band in thesilica column and results from the presence ofp-chlorophenyl-penicillin. This band occupies a position similar to thatin which penicillin K is found in comparable columns. The eluatesobtained from the silica gel sections which make up this uniform bandare combined, cooled to about 0 C., acidified to about pH 2.2 andextracted with three 50 cc. portions of chloroform.

The combined chloroform extracts are then passed through a sflicaadsorption column containing a pH 6.2 phosphate buffer. This silica gelcolumn is developed by percolation with three 100 cc. portions ofchloroform containing successively increasing amounts of methanol in theorder of 1, 2 and 3 percent. The developed silica column is then dividedinto 12 equal sections and each section is eluted with three 30 cc.portions of M/15 phosphate buffer of pH 7.0. Bacteriological assay ofthe eluates shows that about 97 percent of the total antibiotic activityoriginates in a single band in the silica column. The eluates obtainedby extraction of the silica column sections which comprise this band arecombined, cooled to about 0., acidified to about pH 2.2 and extractedwith three 100 cc. portions of ether. The ether extracts are combinedand extracted with about 75 cc. of a cool dilute aqueous solution ofsodium hydroxide to which N/ sodium hydroxide solution is added duringthe course of the extraction so that a final pH of about 7.0 is obtainedin the aqueous phase. From this aqueous solution the sodium salt ofp-chlorophenyl-penicillin may be separated by any suitable means, forexample by freezing and evaporation in vacuo from the frozen state.

-The resulting dry sodium salt of p-chlorophenyl-penicillin is washedwith several portions of anhydrous acetone. It is crystallized bydissolving it in 2 cc. of 90 percent aqueous acetone followed by theaddition of 4 cc. of anhydrous acetone. The salt is recrystallized bydissolving it in 2 cc. of 90 percent aqueous acetone and subsequentlyadding 2 cc. of anhydrous acetone.

The sodium salt of p-chlorophenyl-penicillin prepared according to theabove procedure assayed about 2460 Oxford units per mg. when testedagainst Staph. aureus, strain 209P. A-

diil'erential assay carried out on Staph. aureus, strain 209P, and B.subtilis. strain N. R. R. .L. B-558, gave a value of about 0.73. Theoptical rotation was found to be as a 0.3 percent solution in water.Analysis showed the presence of 49.08 percent carbon, 3.84 percenthydrogen. 7.30 percent nitrogen and 8.85 percent chlorine as comparedwith the calculated values of 49.17 percent carbon, 4.12 percenthydrogen, 7.17 percent nitrogen and 9.07 percent chlorine.

Example 7 The sodium salt of p-chlorophenyl-penicillin may also beprepared in the following manner:

A culture medium may be prepared in the following proportions:

Lactose g 125 Corn steep solids g 150 Calcium carbonate g 25p-Chlorophenylacetic acid g 0.67 Water cc-.. 5,000

12 Example 8 represented by the formula may be prepared in the followingmanner:

A culture medium may be prepared in the following proportions:

Lactose Corn steep solids g Calcium carbonate g 10 N- (2-hydroxyethyl)-p-methylphenylacetamide g. 0.82

ater cc 5,000

The culture medium is placed in a 5 gallon bottle equipped with astirrer and an air inlet tube fitted with an air filter. The culturemedium is sterilized and inoculated with a spore suspension ofPenicillium mold, strain X1612. The

bottle contents are maintained at a temperature of about 23-26 C. andare continuously stirred for five days. Throughout this time, air iscontinuously passed through the air inlet tube. The mold mycelium isthen removed from the aqueous culture medium by filtration and theculture medium is cooled to about 0 C., acidified to about pH 2.2 withorthophosphoric acid and extracted with an equal volume of amyl acetate.The amyl acetate layer is separated and extracted with three 100 cc.portions of cold water to which cold N/10 sodium bicarbonate solution isadded during the course of each extraction until a pH of about 7.1 to7.3 is attained in the aqueous phase. The aqueous extracts are combined,cooled to about 0 0., acidified to about pH 2.2 with orthophosphoricacid and extracted with three 100 cc. Portions of ether. The etherextracts are combined and passed through a chromatographic type silicaadsorption column about 30 mm. in diameter and about 300 mm. long andwhich contains a pH 6.2 phosphate buffer. The silica column is developedby percolation with six 100 cc. portions of ether containingsuccessively increasing amounts of methanol in the order of /2, 1, 1 2,2 and 3 percent.

The developed silica column is divided into about 12 equal sections andeach section is eluted with three 30 cc. portions of M/ 15 phosphatebufl er of pH 7.0. The eluates are assayed bacteriologically todetermine their penicillin content. 100 percent of the total antibioticactivity possessed by all the eluates originates in a single band in thesilica column and results from the presence ofp-methylphenyl-penicillin. The eluates obtained from the silica gelsections which make up this uniform band are combined. cooled to about 0C., acidified to about pH 2.2 with orthophosphoric acid and extractedwith three 50 cc. portions of chloroform. The combined chloroformextracts are passed through a silica adsorption column containing a pH6.2 phosphate buffer. The silica gel column is developed by percolationwith three 100 cc. portions of chloroform containing successivelyincreasing amounts of methanol in the order of 1, 2 and 3 percent. Thedeveloped silica column is then divided into 12 equal sections and eachsection is eluted with three 30 cc. portions of M/15 phosphate buifer ofpH 7.0. Bacteriological assay of the eluates shows 13 band are combined,cooled to about C.,acidifled to about pH 2.2 with orthophosphoric acidand extracted with three 100 cc. portions of ether. The ether extractsare combined and extracted with about 75 cc. of a cold diluteaqueoussolution of sodium hydroxide to which N/10 sodium, 11?- droxide solutionis added during the course of the extraction so that a pH or about 7.0is

obtained in the aqueous phase. From this aque-.

is recrystallized by dissolving it in about 26cc.-

of 87 percent aqueous acetone. followed by the addition of 5.7 cc. ofabsolute acetone.

The sodium salt of p-methy lDhenyl-penicillin prepared according to theabove procedure assayed about 2285 Oxford unitsp'er milligram whentested against staph.-' aureus, strain 209?. A diflere'ntial assaycarried out on Staph. aureus,

strain 209P;'and- B. subtilis, strain N. R. R. L.

3-558 gave a value of about 0.73. Analysis showed the presence of 55.12percent carbon, 5.43percent hydrogen, 7.49 percent nitrogen and 8.56percent sulfur as compared with the calcuiatedvalues of 55.12percentcarbon, 5.17 percent hydrogen, 7.56 percent nitrogen and 8.66percent sulfur. v I

l Example 9 The sodium salt of p-methylphenyl-penicillin may also beprepared in the following manner:

A culture medium is preparedas shown in Example 8 except that in'placeof the 0.82 .g of N- (2'-hydroxyethyl) p methylphenylacetamide there isemployed 2.5 g. of p-methylphenethylamine. 'The sterile culture mediumis placed in a gallon bottle inoculated with a spore suspension ofPenicillium mold, strain X1612. The growth of the mold and the isolationand purification of p-methylphenyl-penicillin arecarried out accordingto the procedure described in Example 8.

Thesodium salt of p methylphenyl-penicillin thus obtained is identicalwith that obtained by the procedure described in Example 8.

' Example 10 The sodium salt of p-nitrophenyl-penicillin represented bythe formula may be prepared in the following manner:

A culture medium may be prepared in the following proportions:

Lactose g 125 Corn steep solids g.. 100 Calcium carbonate g- '10N-p-nitrophenylacetylvaline g 1.05 Water i r 5,000

The culture medium is distributed in 200 cc. portions i111liter-Erlenmeyer flasks, sterilized, inoculated with a spore suspensionof Penicillium mold, strain N. R. R. L. 1976, and stoppered with 5 inthe aqueous phase.

' buffer of pH 7.0. The eluates are assayed bac- 14 cotton pings. Theflasks are maintained at a temperature of about 23-26 C. and shakenconstantly for five. days. The flask contents are then filtered toremove the mold myceiium. the filtrate cooled to about 0 C., acidifiedto about pH 2.2 with orthophosphoric acid and extracted with about 60percent of its volume of amyl acetate. The amyl acetate layer isseparated and extracted with three 100 cc. portions of cold 'watertoiwhich cold N/10 sodium bicarbonate,

solution is added during the course of each extraction until a pH ofabout 7.1 to 7.3 is attained The aqueous extracts are combined, cooledto about 0 C., acidified to about pH 2.2 with orthophosphoric acid andextracted with three 100 cc. portions ofether. The ether;

extracts are combined, and are passed through a chromatographic typesilica adsorption column about 35 mm. indiarneter and 300 mm. long and.

which contains a pH'6.2 phosphate buffer. ,The

silica column is developed bypercolation with six 100 cc. portions ofethercontaining successively increasing amountsof methanol in the orderA, 1, 1%, 2, 2V: and 3 percent,

The developed silica column is divided into about 12.equal sections andeach section'is eluted with three 30 cc. portionsof M/15 phosphateteriologically to determine their penicillincontent. About 38 percent ofthe total antibiotic activity possessed by allthe eluates orignates n asingle band in thesilica column and results from the presence ofp-nitrophenyl-penicillin. This band occupies a position just above thatin which penicillin F is found in comparable columns. The eluatesobtained from the silica gel sections which make up this uniform bandare.v

combined, cooled to about 0 C acidified to about pH 2.2 withorthophosphoric acidand extracted with three cc. portions of chloroform.The combined chloroform extracts are passed through a silica adsorptioncolumn containing a pH 6.2

phosphate bufl'er.- This silica; gel 'columnis developedby percolationwith three 100 cc. portions of chloroform containing successivelyincreasing amounts of methanol in the order of 1, 2 and 3 percent. Thedeveloped silica column is then divided into 12 equal sections and eachsection is eluted with three 30 cc. portions of M/ 15 phosphate bufl'erof pH 1.0. Bacteriological assay of the eluates shows that 100 percentof the total antibiotic activity originates in a single band in thesilica column. The eluates obtained by ex-' traction of the silicacolumn sections whichcomprlse this band are combined, cooled to about 0C.. acidified to about pH 2.2 and extracted with three 100 cc. portionsof ether. The ether e'xtracts are combined and extracted with about 75cc. of cold water to which N/10 sodium bicarbonate solution is addedduring the course of the extraction so that a final pH of about 7.0 isobtained in the aqueous phase. From this aqueous solution the sodiumsalt of p-nitrophenyl-peni cillin may be separated by any suitablemeans, for example, by freezing and evaporation vacuo from the frozenstate.

The dry amorphous sodium salt of p-nitrophenyl-penicillin is treatedwith 2 cc. of absolute acetone in which it almost completely dissolves.

but from which it precipitates in crystalline form upon standing abouttwo hours. The mixture is centrifuged and the solid washed with severalportions of absolute acetone. The salt is redissolved in 1.6 cc. ofpercent aqueous acetone and reprecipitated by'the addition of 6 cc. of

auaaoo 15 absolute acetone. A further recrystallization is eil'ected bysolution of the salt in 0.3 cc. of. 90 percent acetone followed by theaddition of a total of -3 cc. of absolute acetone added in portions overa period of about four hours.

The sodium salt of p-nitrophenyl-penicillin prepared according to theabove procedure assayed about 1640 Oxford units per-milligram whentested against Staph. aureus, strain 209?. A difierential assay carriedout on Staph. aureus, strain 209P, and B.. subtilis, strain N. R. R. L.

B-558, gave a value of about 0.84. The ultra violet absorption spectrumshowed a characteristic peak at about 270 m indicative of the pres- Ionce of the p-nitrophenyl group.

Example 11 The sodium salt. of p-nitrophenyl-penicillin may also beprepared in thefollowing manner:

A culture medium is prepared as shown in Example 10 except that in placeof the 1.05 g. of N- p-nitrophenyl-acetylvaline there is employed 1.05g. of N-p-nitrophenyl-acetylisoleucine. The culture medium may then-'be' treated substantially according to the procedure described inExample 10, and the penicillin produced in the culture medium'may beisolated by the procedure substantially as described in Example 10.

1 The sodimn salt of -p-nitrophenyl-penicillin thus prepared isidentical to p-nitrophenyl-penicillln sodium salt prepared according tothe pro cedure described in Example 10.

Example 12 The sodium salt of p-fluorophenyl-penicillin represented bythe formula FQCufinNIOE B may be prepared in the following. manner:

A culture medium may be prepared in the following proportions: I

Lactose V g..- 125 Com steep solids g 100 Calcium carbonate -g 10 N (2hydroxyethyl) -p-fluorophenylacetamide I g 0.72 Water cc 5,000

The. culture medium is distributed in 200 cc. portions in 1 literErlenmeyer flasks, sterilized, inoculated with a spore suspension ofPenicillium mold, strain N. R. R. L 1976, and stoppered with cottonplugs. The flasks are maintained at a temperature of about 23-26 C. andshaken constantly for five days. The flask contents are then filtered toremove the mold mycelium, the filtrate cooled to about 0., acidified toabout pH 2.2 with orthophosphoric acid and extracted with an equalvolume of any] acetate. The amyl acetate layer is separated andextracted with three 100 cc. portions of cold water to which cold N/sodium bicarbonate solution is added during the course of eachextraction until a pH of about 7.1 to 7.3 is attained in the aqueousphase. The aqueous extracts are combined, cooled to about 0 0.,

acidified to about pH 2.2 with orthophosphoric acid and extracted withthree 100 cc. portions of ether. The ether extracts are combined. andare passed through a chromatographic type silica adsorption column about30 mm. in diameter and 'thisbandare acid and extracted The resulting Thedeveloped silica about l2-equalsections and each section is eluted withthree 30 cc. portions of I/l5 phosphate buffer of PH 7.0. Theeillatesare assayed bacteriologically to determine their penicillincontent. Practically all of the total antibioticactivity possessed byall the eluates originates in a single band in the silica column andarisesi'rom the presence of p-fluorophenyl-penicillin. The eluatesobtained from the silica gel sections which make up this uniform bandare combined. cooled to about 0 C., acidified to about pH 2.2with'orothphosphoric acid and extracted with three 500 cc. portions ofchloroform. The combined chloroform extracts are passed through a silicaadsorption column containing "a pH 6.2 phosphate buffer. This silica gelcolilnn is developed by percolation with three 100 cc. portions ofchloroform containing successively increasing amounts of methanol in theorder of l, 2 and 3 percent. The developed silica column is then dividedinto 12 equal sections and each section iseluted with three 30 cc.portions oi M/lfi phosphate buifer of pH 7.0. Bacteriological assayoi'the eluates shows that almost 100 percent of the total antibioticactivity originates in a single band in'the silica column. The eluatesobtained by extraction of the silica column sections which comprisecombined, cooled to about 0'' C., acidified ,to about pH 2.2 withorthophosphorie with three 100 cc. portions of ether. The ether extractsare combined and extracted with about 75 cc. of a cold dilute aqueoussolution of sodium hydroxide to which N/l0 sodium hydroxide solution isadded during the course of the extraction at 7.0 is obtained in theaqueous phase. From this aqueous solution the sodium salt ofp-iluorophenyl-penicillin may be able means. for example by freezing andevaporation in vacuo from the from state.

I dry sodium salt of p-iluorophenyl-penicillin is washed with severalportions of anhydrous acetone. It may be crystallized by pfluorophenyl-peniclllin ferential assay :when carried out on Staph.

aureus, strain 209?, and B. subtilis, strain N. R.

R. L. B-558, gave a-v'alue of-about 0.89. Analysis of a sample 01p-iluorophenyl-penicillin showed the presence of 51.27 percent carbon,4.15 percent ,drogen, 7.49 percent nitrogen and 8.43 percent sulfur ascompared with the calculated values of 4 51.33 percent carbon, 4.31percent hydrogen, 7.47

percent nitrogen and 8.50 percent sulfur;

Example 13 I I a The sodium salt-of p-iluorophenyl-penicillin may alsobe prepared in the following manner:

A culture medium may be prepared in the following proportions: Y

Lactose g Corn steep solids g- Calcium carbonate g 25p-Fluorophenethylamine g 2.0 Water cc 5,000

The sterile culture medium is placed in a 5 gallon bottle equipped witha stirrer and an air inlet tube fitted with an air filter. The culturecohnnn is divided into so that a final pH of.

separated by any suit- Oxford units per mg. when 7 medium is inoculatedwith a spore suspension of Penicillium mold, strain X1612. The bottlecontents are maintained at a temperature of about 23-26 C., and arestirred continuously for five days. Throughout this time air iscontinuously passed through the air inlet tube. The mold mycelium isthen removed from the aqueous culture medium by filtration. The desiredpiluorophenyl-penicillin may be isolated and purified by substantiallythe same procedure as described'in Example 12.

The sodium salt of p-fluorophenyl-penicillin thus prepared is identicalwith p-fluorophenylpenicillin sodium salt prepared according to theprocedure described in Example 12.

Example 14 The sodium salt of o-fluorophenyl-penicillin represented bythe formula may be prepared in the following manner:

A culture medium may be prepared in the following proportions:

Water cc 5,000

The sterile culture mediumis inoculated with mold spores and subsequentmold growth and isolation of o-fiuorophenyl-penicillin is effected bysubstantially the same procedure as described in Example 12.

The dry amorphous sodium salt of o-fluorophenyl-penicillin isrecrystallized by solution in 1 /2 cc. of 90 percent aqueous acetonefollowed by the addition of 2 /2 cc., of absolute acetone which is addedslowly and with shaking. Recrystallization may be eflected by dissolvingthe penicillin in 90 percent aqueous acetone and precipitation withabsolute acetone.

A sample of the sodium salt of o-fluorophenylpenicillin was found topossess a value of about 1340 Oxford units per milligram when testedagainst Staph. aureus, strain 209?. A differential assay carried out onStaph. aureus, strain 209P, and B. subtilz's, strain N. R. R. L. 3-558,gave a value of about 1.1. Analysis showed the presence of 51.93 percentcarbon, 4.59 percent hydrogen, 7.81 percent nitrogen and 8.09 percentsulfur as compared with the calculated values of 51.33 percent carbon,4.29 percent hydrogen, 7.49 percent nitrogen, and 8.56 percent sulfur.

Example The sodium salt of m-fluorophenyl-penicillin represented by theformula may be prepared in the following manner:

A culture medium may be prepared in the following proportions:

The sterile culture medium is inoculated with mold spores, andsubsequent mold growth and isolation of m-fluorophenyl-penlcillin iseffected by substantially the same procedure as described in Example 12.

The dry amorphous sodium salt of m-fluorophenyl-penicillin may berecrystallized and purlfled by solution in 90 percent aqueous acetonefollowed by precipitation with absolute acetone.

A sample of the sodium salt of m-fluorophenylpenicillin was found topossess a. value of about 2340 Oxford units per milligram when testedagainst Staph. aureus, strain 209P. A differential assay carried out onStaph. aureus, strain 209P, and B. subtilis, strain N. R. R. L. 3-558,

1 gave a value of about 0.76. Analysis showed the presence of 51.47percent carbon, 4.19 percent hydrogen, 7.61 percent nitrogen and 8.21percent sulfur as compared with the calculated values of is attained inthe aqueous phase.

51.33 percent carbon, 4.31 percent hydrogen, 7.49 percent nitrogen and8.56 percent sulfur.

Example 16 The sodium salt of p-bromophenyl-penicillin represented bythe formula BIQ-Ommmsrn may be prepared in the following manner.

The culture medium is distributed in 200 cc. portions in 1 literErlenmeyer flasks, sterilized, inoculated with a spore suspension ofPenicillium mold, strain N. R. R. L. 1976, and stoppered with cottonplugs. The flasks are maintained at a temperature of about 23-26" C. andshaken constantly for five days. The flask contents are then filtered toremove the mold mycelium, the filtrate cooled to about 0 C., acidifiedto about pH 2.2 with orthophosphoric acid and extracted with an equalvolume of amyl acetate. The amyl acetate layer is separated andextracted with three 100 cc. portions of cold water to which cold N/ 10sodium bicarbonate solution is added durin the course of each extractionuntil a pH of about 7.1 to 7.3 The aqueous extracts are combined, cooledto about 0 C., acidified to about pH 2.2 with orthophosphoric acid andextracted with three 100 cc. portions of ether. The ether extracts arecombined and are passed through a chromatographic type silica adsorptioncolumn about 30 mm. in diameter and 300 mm. long and containing a pH 6.2phosphate buffer. The silica column is developed by percolation with six100 cc. portions of ether containing successively increasing amounts ofmethanol in the order of 1, 1 2, 2 /2 and 3 percent.

The developed silica column is divided into about 12 equal sections andeach section is eluted with three 30 cc. portions. of M/ 15 phosphatebufier of pH 7.0. The eluates are assayed bacteriologically to determinetheir penicillin content. 100 percent of the total antibiotic activitypossessed by all the eluates originates in a single band in the silicacolumn and arises from the presence of p-bromophenyl-penicillin. Thisband occupies a position slightly below that in which penicillin G isfound in comparable columns. The eluates obtained from the silica gelsections which make up this uniform band are combined, cooled to aboutC., acidified to about pH 2.2 and extracted with three 50 cc. portionsof chloroform. The combined chloroform extracts are passed through asilica adsorption column similar to that used before. This silica gelcolumn is developed by percolation with three 100 cc. portions ofchloroform containing successively increasing amounts of methanol in theorder of 1, 2 and 3 percent. The developed silica column is then dividedinto 12 equal sections and each section is eluted with three 30 cc.portions of M/ 15 phosphate bufier of pH 7.0. Bacteriological assay ofthe eluates shows that about 100 percent of the total antibioticactivity originates in a single band in the silica column. The eluatesobtained by extraction of the silica column sections which comprise thisband are combined, cooled to about 0 C., acidified to about pH 2.2. withorthophosphoric acid and extracted with three 100 cc. portions of ether.The ether extracts are combined and extracted with about 75 cc. of acold dilute aqueous solution of sodium hydroxide to which N/l0 sodiumhydroxide solution is added during the course of the extraction so thata final pH of about 7.0 is obtained in the aqueous phase. From thisaqueous solution the sodium salt of p-bromophenyl-penicillin may beseparated by any suitable means, for example, by freezing andevaporation in vacuo from the frozen state.

The dry sodium salt of p-bromophenyl-penicillin is treated with 2 cc. ofabsolute acetone from which the crystalline salt separates afterstanding for about one hour. The salt may be recrystallized by treatmentof a solution of the salt in 90 percent aqueous acetone with an excessof absolute acetone.

The sodium salt of p-bromophenyl-penicillin assayed about 2460 Oxfordunits per mg. when tested against Staph. aureus, strain 209P. Adifierential assay carried out on Staph. aureus, strain 209P, and B.subtz'lis,-strain N. R. R. L. 3-558, gave a value of about 0.65.Analysis showed the presence of 44.36 percent carbon, 3.93 percenthydrogen, 6.55 percent nitrogen and 16.91 percent bromine as comparedwith the calculated values of 44.14 percent carbon, 3.71 percenthydrogen, 6.05 percent nitrogen and 17.25 percent bromine.

Example 17 The sodium salt of p-bromophenyl-penicillin may also beprepared in the following manner:

A culture medium may be prepared in'the following proportions:

Lactose g 125 Corn steep solids g 150 Calcium carbonate g 25fl-p-Bromophenethanol -g, 1.0 Water cc 5,000

Example 18 The sodium salt of phenoxy-penicillin represented by theformula @mwmwwsm may be prepared in the following manner:

A culture medium may b prepared in the following proportions:

Lactose g 125 Corn steep solids ..g 150 Calcium carbonate g 25 N (2hydroxyethyD-phenoxyacet amide ..g 0.78 Water cc 5,000

The culture medium is distributed in 200 cc. portions in 1 literErlenmeyer flasks, sterilized, inoculated with a spore suspension ofPenicillium mold, strain N. R. R. L. 1976, and stoppered with cottonplugs. Growth of the mold and isolation of the sodium salt of thephenoxy-penicillin may be effected by the procedure described in Example1.

The sodium salt thus obtained is purified by dissolving it in 2 cc. ofabsolute acetone from which upon standing it separates in crystallineform. It is separated by centrifugation and washed with small portionsof absolute acetone. It is then dissolved in about 3 cc. of percentaqueous acetone, the solution filtered and 4 cc. of absolute acetoneadded to the filtrate'whereupon the pure crystalline material separates.The salt is recrystallized by dissolving it in about 3 cc. of 85 percentaqueous acetone followed by the addition of about 7 cc. of absoluteacetone.

The sodium salt of phenoxy-penicillin assayed about 1660 Oxford unitsper milligram when tested against Staph. aureus, strain 209P. Adifferential assay when carried out on Staph. aureus, strain 209P, andB. subtilis, strain N. R. R. L. 3-558, gave a value of about 0.87.Analysis showed the presence of 51.17 percent carbon, 4.49 percenthydrogen, 7.59 percent nitrogen and 8.50 percent sulfur as compared withthe calculated values of 51.34 percent carbon, 4.60 percent hydrogen,7.55 percent nitrogen and 8.61 percent sulfur.

Example 1.9

The sodium salt of phenoxy-penicillin may also be prepared as follows:

The culture medium as described in Example 18 is inoculated withPe'nicillium mold, strain X1612, and growth of the mold is effected in afive gallon bottle equipped with a stirrer and an air inlet tube fittedwith an air filter as described in Example 8. The isolation andpurification is effected according to the procedure described in Example18.

The sodium salt of phenoxy-penicillin thus prepared is identical withphenoxy-penicillin sodium salt prepared according to the proceduredescribed in Example 18.

Example 20 The sodium salt of p-iodophenyl-penicillin represented by theformula IQGI-HIMOiBNs may be prepared in the following manner:

A culture medium may be prepared in the following proportions:

Lactose g 125 Corn steep solids g Calcium carbonate g 10 N (2hydroxyethyl) p iodophenylacetamide g-.. Water or The culture medium isdistributed in 200 cc. portions in 1 liter Erlenmeyer flasks,sterilized, inoculated with a spore suspension of Penlcillium mold,strain X1612, and stoppered with cotton plugs. The flasks are maintainedat a temperature of about 23-26 C. and shaken constantly for flve days.The flasks contents are then filtered to remove the mold mycelium. Thep-iodophenylpenicillin present in the filtrate may be isolated accordingto the procedure described in Example 1.

The dry amorphous sodium salt of p-iodophenyl-penicillin obtained byevaporation of the aqueous solution or the sodium salt from the frozenstate assayed about 27,70 Oxford units per milligram when tested againstStaph. aureus, strain 209P. A differential assay carried out on Staph.aureus, strain 209?, and B. subtilis, strain N. R. R. L. 3-558, gave avalue of about 0.67.

Example 21 The sodium salt of p-tolylmercapto-penicillin represented bythe formula maybe prepared in the following manner:

A culture medium may be prepared in the following proportions:

Iactose g 125 Corn steep solids g 100 Calcium carbonate gN-p-tolylmercaptoacetylvaline g 1.0 Water cc.. 5,000

The culture medium is distributed in 100 cc. portions in 1 literErlenmeyer flasks, sterilized, inoculated with a spore suspension ofPenicillium mold, strain X1612, and stoppered with cotton plugs. Theflasks are maintained at a temperature of about 23-26 C. and shakenconstantly for flve days. The flasks contents are then filtered toremove the mold mycelium. The p-tolylmercapto-penicillin present in thefiltrate may be isolated according to the procedure described in Example1.

The dry amorphous sodium salt of p-tolylmercapto-penicillin obtained byevaporation of the aqueous solution of the sodium salt from a frozenstate is dissolved in 1.5 cc. of absolute acetone and the solutionfiltered to remove inorganic material. Upon addition of an excess ofanhydrous ether to the filtrate, the sodium salt of-p-tolylmercapto-penicillin is precipitated as an oil. The oil istreated with an equal volume of absolute acetone and allowed to stand inthe refrigerator for about 12 hours. It is then transferred to acrystallizing dish and placed in a vacuum desiccator. After standing forsome hours the salt crystallizes. The solid material is treated with 1cc.

of absolute acetone from which, upon standing at room temperature andwith occasional scratching with a glass rod, the sodium salt separatesin crystalline form.

The sodium salt of p-tolylmercapto-penicillin thus obtained assayedabout 1285 Oxford units per milligram when tested against Staph. aureus,strain 209P. A differential assay carried out on Staph. aureus, strain209P, and B. subtilis, strain N. R. R. L. B-558, gave a value of about0.83.

Example 22 The sodium salt of cyclohexyl-penicillin represented by theformula CHr-CHI C CH-GMHHNIOSNB CHr-C I may be prepared in the followingmanner:

A culture medium may be prepared in the following proportions:

Lactose g 125 Corn steep solids g Calcium carbonate g 10N-cyclohexylacetylvaline g 1.0 Water cc 5,000

crystalline sodium salt is isolated by centrifugation and washed withsmall portions of anhydrous acetone. It is dissolved in about 0.5 cc. of92 percent aqueous acetone, the solution is filtered and the filtrate istreated with 5 cc. of absolute acetone whereupon the crystalline sodiumsalt of cyclohexyl-penicillin precipitates. The sodium salt ofcyclohexyl-penicillin prepared according to the above procedure assayedabout 1700 Oxford units per milligram when tested against Staph. aureus,strain 209P. A difierential assay carried out on Staph. aureus, strain209?, and B. subtilis, strain N. R. R. L. 3-558, gave a value of about0.79. Other examples of new penicillins produced substantially inaccordance with the procedure described in the preceding examples aretabulated below together with the precursor compound used in theproduction of the penicillin and the dififer-. ential assay value of thepenicillin produced.

Penicillin Precursor Compound f gg gfig 3, i dichlorophenyl-penicillinN-Ef-hydroxyethyl)-3,4-dichlorophenyl-acetamide 0.64o-bromophenyl-pemclllin N- 2-hydroryethyD-o-bromophenyl-acetamide 0.625,6,7,8-tetrahydro-2-naphthylpenicrllinN-(iihydroxyethyl)-5,6,7,8-tetral1ydro-2-naphthylaceta- 0.73 v m e.

23 I Additional examples of new penicillins which may be produced inaccordance with this invention are set forth below, together with theprecursor compounds used in their production.

24 mixture is dissolved in about 50 cc. of ether-petrm leum ethermixture and the solution is partially evaporated to yield crystals of N-(Z-hydroxyethyl) -p-methoxyphenylacetamide. The crystals are PenicillinPrecursor Compound athylmeroapto-penicillin ln-methoXyphenyl-pemcillir ip-methoxyphenoxy-pemcillln m-trifluoromethyl henylnicillinp-benzyloxypheny nic' lin.. p-naphthoxynici in- 6-iluoro-2-napthyl-penicilhm. 6-bromo-2-naphthyl-penicillin.2,4-dichlorophenyl-peniciilin p-phenylmercaptophenyl-penicill'p-naphthylmermptopenicillin...- p-allyloxyphenyl-penicillinp-methylphenoxy-penicillin m-trliluoromethylphenoxyni3,4-dlmethylphenylnicillm phenylmercaptomet yl-filenicillin.m-hydroxyphenyl-penic in p-aminophenyl-penicillm styryl-penicillinp-carbethoxyhydroxy henyl-penicillin. Q-cyclope'ntenyl-penicfilin fl-mhthyI-penicillln rophenyl-penicillin m-chlorophenylnicillinm-methy phonypenicillinp-chlorophenoxy-penicillin m-nitrophenyl-penicillinm-bromoghenyl-penlcillin 2,3-dimet oxyphenyl-penicillin.o-methoxy-z-naphthyl-penicillin ad dlbromophenyl-pemcillim3-chloro-4-bromophenylp-phenoxypheny -penic' in--."p-cyanophenyl-penicillin zzz 22 22 z -hydroxyeth fiydroxyeth4-dibromophenylacetamida.

For the purposes of convenience the preparations of certain novelcompounds useful in carrying out this invention are given below.

Preparation of N -(2-hydr0:ruethyl) -p-iodophenylacetamideN-(2-hydroxyethyl) p iodophenylacetamide represented by the followingformula rQcmlLmr-omcmon may be prepared in the following manner:

A mixture of 16 g. of methyl p-iodophenylacetate and 4.7 g. ofethanolamine are heated together at about 100 C. for about 12 hours. Thereaction mixture is then dissolved in about 200 cc. of boiling ethylacetate, treated with decolorizing carbon, filtered, and the filtrateevaporated to a volume of about 100 cc. Upon cooling the concentratedsolution, preferably to a temperature below 5 C.,N-(Z-hydroxyethyl)-p-iodophenylacetamide precipitates in crystallineform. It is purified by recrystallization from a mixture of ethylacetate and petroleum ether.

N-(Z-hydroxyethyl) p iodophenylacetamide thus prepared melted at about112-113 C. Analysis showed the presence of 39.32 percent carbon and 4.01percent hydrogen as compared with the calculated values of 39.36 percent carbon and 3.97 percent hydrogen;

Preparation of N-(Z-hydroxyethyl) -p-metho:tz phenylacetamide N-(2hydroxyethyl) p methoxyphenylacetamide represented by the followingformula 0 cmoQcmt-mr-cmcmon may be prepared in the following manner:

A mixture of 5.4 g. of methyl p-methoxyphenylacetate and 1.9 g. ofethanolamine is heated at about 140 C. for about 12 hours. The reactionfurther purified by recrystallization from ethylene dichloride.

N-(2 hydroxyethyl) p methoxyphenylacetamide thus prepared melts at about86-88 C. Analysis showed the presence of 6.64 percent nitrogen ascompared with the calculated value of 6.69 percent.

Preparation of N-(2-h1 droz1/ethyl) -athienylacetamideN-(2-hydroxyethyl)--thienylaceta.mide represented by the followingformula noon o a a H H -oH.c-NH-cmorr,0n

may be prepared in the following manner:

A mixture of 7.34 g. of methyl a-thienylacetate and 3.1 g. ofethanolamine is heated at about 135 C. for about 11 hours. The excessethanolamine is then removed from the reaction mixture by heating thelatter at about C. under reduced pressure until all the ethanolamine isremoved. N (2 hydroxyethyl) --thienylacetamide is obtained incrystalline form upon prolonged standing at room temperature.

N-(2 hydroxyethyl) -a-thienylacetamide thus prepared melted at about66--6'l C. Analysis showed the presence of 7.96 percent nitrogen ascompared with the calculated value of 7.58 percent.

Preparation of N-p-chloropherwlacetylvaline N-p-chlorophenylacetylvalinerepresented by the following formula may be prepared in the followingmanner:

p-Chlorophenylacetyl chloride is prepared by treating 8.5 g. ofp-chlorophenylacetic acid with 15 cc. of thionyl chloride, allowing themixture to stand at room temperature for about 12 hours and thenremoving the excess thionyl chloride in vacuo. The resultingp-chlorophenyiacetyl chloride is dissolved in about 30 cc. of anhydrousether and the solution added in small portions and with stirring to asolution of 6.5 g. of valine dissolved in 110 cc. of 1 N sodiumhydroxide. Stirring is continued for two hours after which time the.aqueous layer is separated from the supernatant etherlayer, acidifiedand cooled to about C., whereupon N-p-chlorophenylacetylvaline issubstantially completely precipitated. The N-p-chlorophenylacetylvalineis purified by recrystallization from aqueous alcohol.

N-p-chlorophenylacetylvaline thus prepared melted at about 144-145 C.Analysis showed the presence of 5.0 percent nitrogen as compared withthe calculated value of 5.19 percent.

Preparation of N-(Z-hydroryethyl) -ofluorophenylacetamide N (2-hydroxyethyl) -o-fluorophenylacetamide represented by the followingformula Preparation of N-(Z-hydroxyethyl) -mfluorophenylacetamide N-(2-hydroxyethyl) -m-fluorophenylacetamide represented by the followingformula 0 ll @cnm-1vrr-cmcmon may be' prepared from ethylm-fluorophenylacetate and ethanolamine by substantially the same methodas used in the preparation of N-(2- hydroxyethyl)-o-fiuorophenylacetamide.

N (2-hydroxyethyl) -m-fluorophenylacetamide thus prepared melted atabout '15-'77" C. Analysis showed the presence of 7.10 percent nitrogenas compared with the calculated value of 7.10 percent.

Preparation of N-(Z-hydroxyethyl) -pfluorophenylacetamtde N (2hydroxyethyl) -p-fluorphenylacetamide represented by the followingformula may be prepared from methyl p-fluorophenylacetate andethanolamine by substantially the same method as used in the preparationof N-(2- hydroxyethyl) -o-fluorophenylacetamide. I

N (2-hydroxyethy1) -p-fluorophenylacetamide thus prepared melted atabout 73-74" C. Analy- 26 sis showed the presence of 7.03 percentnitrogen as compared with the calculated value of '1.10 percent.

Preparation of N-(Z-hydrozyethyl) {l -methylphenylacetamide N (2hydroxyethyl) p methylphenylacetamide represented by the formula 0moQ-omE-nn-cmcmon Preparation of N-y-(p-bromophen'yl) -butyrylvalineN-q- (p-bromophenyl) -butyrylvaline represented by the following formulaOOH may be prepared in the following manner:

-(p-bromophenyl) --butyryl chloride is prepared by reacting 23.5 g. ofY-p-bromophenylbutyric acid with 30- cc. of thionyl chlorideat v roomtemperature for about 15 hours. The excess thionyl chloride is removedin vacuo. The residue comprising Y-(p-bromophenyl) -butyryl chloride, isdissolved in about 60 cc. of ether and the solution is added in smallportions and with stirring to a solution of 12 g. of valine dissolved in200 cc. of 1 N sodium hydroxide. Stirring is continued for two hoursafter which period of time theaqueous layer is separated from thesupernatant ether layer, acidified and cooled to about 0 C., whereuponN- y-(p-bromophenyl) -butyrylvaline is substantially completelyprecipitated. It is purified by recrystallization from a mixture ofmethl acetate and petroleum ether.

N-y-(p-bromophenyl) -butyrylvaline thus prepared melted at about 134-135C. Analysis showed the presence of 4.11 percent nitrogen as comparedwith the calculated value of 4.21 percent.

I Preparation of N-p-nitrophenylacetyloaline N-p-nitrophenylacetylvalinerepresented by the following formula I o c omQouiPi-mnm COOKN-p-nitrophenylacetylisoleucine represented by the following formula OOHCalls may be prepared in the following manner:

g. of p-nitrophenylacetyl chloride is dissolved in about cc. of etherand the solution added to a solution of 8 g. of isoleucine dissolved inabout 60 cc. of 6 N sodium hydroxide solution. The mixture is shaken forabout 10 minutes and cooled to below 10 C. The aqueous layer isseparated from the ether layer and acidified with dilute hydrochloricacid whereupon substantially all of the N-p-nitrophenylacetylisoleucineprecipitates. It is purified by recrystallization from alcohol.

N-p-nitrophenylacetylisoleucine thus prepared melted at about 113-115 C.Analysis showed the presence of 9.65 percent nitrogen as compared withthe calculated value of 9.65 percent nitrogen.

Preparation of N-(Z-hydroxyethyl) -phenoa:y-

acetamide N-(2-hydroxyethyl) -phenoxyacetamide represented by thefollowing formula 0 ll Q0 CHM-NHCHaCHzOH may be prepared in thefollowing manner:

Methyl phenoxyacetate is prepared by esterification of phenoxyaceticacid with methyl alcohol and sulfuric acid. 4.65 g. of the methylphenoxyacetate and 1.53 g. of ethanolamine are heated at 150 C. forabout two hours. Excess ethanolamine is then removed by subjecting thereaction mixture to a vacuum while maintaining the temperature of thereaction mixture at about 150 C. Upon cooling the residue comprisingN-(2- hydroxyethyl) phenoxyacetamide crystallizes- It is purified byrecrystallization from a mixture of ethyl acetate and petroleum ether.

N-(2 hydroxyethyl) phenoxyacetamide thus prepared melted at about 4548C.- Analysis showed the presence of 7.15 percent nitrogen as comparedwith the calculated value of 6.91 percent.

If'reparation of N-allyl-p-chloropropio'namideN-allyl-fi-chloropropionamlde represented by the formula Preparation ofN-p-tolylmercaptoacetzllvaline N-p-tolylmercaptoacetylvaline representedby the formula CH; CH:

may be prepared as follows:

p-Tolylmercaptoacetic acid is prepared by heating a solution of 59 g. ofp-thiocresol, cc. of 12.5 N sodium hydroxide solution, 50.3 g. ofchloroacetic acid and 1 liter of water for two hours at 8090 C. Thereaction mixture is then OOH " cooled and acidified with dilutehydrochloric acid whereupon the p-tolylmercaptoacetic acid isprecipitated. It is purified by recrystallizing it from a mixture ofether and petroleum ether. p-Tolylmercaptoacetic acid thus preparedmelted at about -86 C. Analysis showed the presence of 59.20 percentcarbon and 5.19 percent hydrogen as compared with the calculated valuesof 59.31 percent carbon and 5.53 percent hydrogen.

3.64 g. of p-tolylmercaptoacetic acid is converted to the correspondingacid chloride by treatment with 20 cc. of thionyl chloride. The reactionmixture is maintained at room temperature for about 12 hours and theexcess thionyl chloride is then removed in vacuo. The resultingp-tolylmercaptoacetyl chloride is converted toN-p-tolylmercaptoacetylvaline by reacting the acid chloride with 3.5 g.of valine according to the procedure described in the preparation ofN-y-(p-bromophenyl)-butyrylva.1ine. The N-ptolylmercaptoacetylvaline ispurified by recrystallization from a mixture of ethanol, ether andpetroleum ether. N-p-tolylmercaptoacetylvaline thus prepared melted atabout 136-138" C. Analysis showed the presence of 4.95 percent nitrogenas compared with a calculated value of 4.97 percent nitrogen.

Preparation of N- (Z-hydromyethyl) -obromophenylacetamide N-(Z-hydroxyethyl) -o-bromophenylacetamide represented by the formula maybe prepared by reacting 51 g. of ethyl obromophenylacetate and 5 g. ofethanolamine according to the method described in the preparation ofN-(2-lwdroxyethyl) -p-methoxyphenylacetamjde. N- (2-hydroxyethyl)-o-bromopheny1- acetamide thus prepared and recrystallized from ethylenedichloride melted at about 106-107 C. Analysis showed the presence of5.51 percent nitrogen as compared with the calculated value of 5.43percent nitrogen.

Preparation of N-ethylmercaptoacetylvaline N-ethylmercaptoacetylvalinerepresented by the formula may be prepared as follows:

Ethylthioglycolic acid chloride is prepared by reacting 12 g. ofethylthioglycolic acid with 25 cc. of thionyl chloride at roomtemperature for about 12 hours. The excess thionyl chloride is thenremoved in vacuo and the resulting ethyl- Preparation of N- (2'-h1ldro:rilethyl)- 3,4-dichlorophenylacetamide N- (2 -hydroxyethyl)-3,4-dichlorophenylacetamide represented by the formula ImQOEd-NH-CmCmOH may be prepared as follows:

161 g. of 3,4-dichlorotoluene is heated to 150- 160 C. and 160 g. ofbromine added slowly. The resulting 3,4-dichlorobenzyl bromide is mixedwith 70 g. of potassium cyanide, 400 cc. of ethanol and 1000 cc. ofwater and the mixture refluxed with stirring for about five hours. Thealcohol is removed from the reaction mixture in vacuo and the oily layerof 3,4-dichlorobenzonitrile is separated and hydrolyzed by refluxingwith a mixture of 400 cc. of concentrated sulfuric acid and 300 cc. ofwater. The reaction mixture is cooled whereupon the3,4-dichlorophenylacetic acid formed during the hydrolysis precipitates.The precipitate is dissolved in dilute sodium hydroxide solution, thesolution filtered and the filtrate acidified with dilute hydrochloricacid whereupon the 3,4-dichlorophenylacetic acid is precipitated. Theacid is then esterified by refluxing it with 600 cc. of absolute ethanoland cc. of concentrated sulfuric acid for about 4 hours. The bulk of thealcohol is removed in vacuo and the residue poured into cold water. Thelayer of ethyl 3,4-dichlorophenylacetate is separated and the esterpurified by distillation in vacuo. Ethyl 3,4-dichlorophenylacetate thusprepared boiled at3112-115 C. at 0.2 mm. pressure. Analysis showed thepresence of 51.78 percent carbon and 4.42 percent hydrogen as comparedwith the calculated values of 51.50 percent carbon and 4.32 percenthydrogen.

45 g. of ethyl 3,4-dichlorophenylacetate and 13 g. of ethanolamine areheated at about 125 C. for hours. The reaction mixture is then heated invacuo to remove the excess ethanolamine. The residue ofN-(2'-hydroxyethyl)- 3,4-dichlorophenylacetamide is recrystallized fromethylene dichloride. N-(2'-hydroxyethyl)- 3 4 dichlorophenylacetamidethus prepared melted at about 113-114 C. Analysis showed the presence of5.79 percent nitrogen as compared with the calculated value of 5.64percent nitrogen.

Preparation of N- (2'-hydr0:cyethyl) -5,6,7,8-

tetrahydronaphthyl-Z-acetamide N (2'-hydroxyethyl) -5,6,'7,8-tetrahydronaphthyI-Z-acetamide represented by the formula e 0 IIcmc-Nn-omcnmn may be prepared as follows:

50 g. of 2-acetotetralin, 13 g. of sulfur and 40 cc. of morpholine arerefluxed for about 15 hours.

To the reaction mixture is then added a mixture 300 cc. of water andrefluxing is continued for 15 hours. The reaction mixture is thencooled, and extracted with ether. The ether extract is separated and isextracted with dilute sodium hydroxide solution. The extract of aqueousalkali is separated, acidified with dilute hydrochloric acid and cooledto about 0 C. whereupon 5,6,7,8-tetrahydro-2-naphthylacetic acidprecipitates. The acid is separated and esterified by refluxing it with600 cc. of absolute ethanol and 10 cc. of concentrated sulfuric acid fora period of about 5 hours. The bulk of the alcohol is then removed invacuo and the residue is poured into cold water. The layer of ethyl5,6,7,8-tetrahydro-2-naphthylacetate is separated and the ester purifiedby distillation in vacuo. Ethyl 5,6,'7,8-tetrahydro-2-naphthylacetatethus prepared boiled at -143 C. at 0.5 mm. pressure. Analysis showed thepresence of 76.84 percent carbon and 8.62 percent hydrogen as comparedwith the calculated values of 77.03 percent carbon and 8.31 percenthydrogen.

23 g. of ethyl 5,6,7,8-tetrahydro-2-naphthylacetate and 8 g. ofethanolamine are reacted at 125-130 C. for about 15 hours. The reactionmixture is then heated to about C. in vacuo to remove the excessethanolamine and the residue comprising N-(2-hydroxyethyl)-5,6,'l,8-tetrahydro-2-naphthylacetamide is purified by recrystallization fromethylene dichloride. N (2'-hydroxyethyl)-5,6,7,8-tetrahydro-2-naphthylacetamide thus prepared melted at about88-90 C. Analysis showed the presence of 6.19 percent nitrogen ascompared with the calculated value of 6.00 percent nitrogen.

Preparation of N- (Z-hydroxyethyl) -m-methoa:y-

phenylacetamide N (Z-hydroxyethyl) -m-methoxyphenylacetamide representedby the formula may be prepared from 4'7 g. of ethylm-methoxyphenylacetate and 16 g. of ethanolamine according to the methoddescribed in the preparation of N(2-hydroxyethyl)-p-methoxyphenylacetamide. N .(2-hydroxyethyl)-m-methoxyphenylacetamide thus prepared melted at about 58 59 C.Analysis showed the presence of 6.68 percent nitrogen as compared with acalculated value of 6.69 percent nitrogen.

Preparation of N-(Z-hydroxyethyl) -m-trifluoromethylphenylacetamide N (2hydroxyethyl) m trifluoromethylphenylacetamide represented'by theformula during the addition. The mixture is distilled with steam and them-trifluoromethylbenzonitriie is separated from the aqueous distillateby extraction with ether. The ether extract is dried, the etherevaporated and the residue comprising m-trifluoromethylbenzonitrile ispurified by distillation. m-Trifluoromethylbenzonitrile thus preparedexhibited a boiling point of about 188- 190 C. at atmospheric pressure.Analysis showed the presence of 8.05 percent nitrogen as compared with acalculated value of 8.19 percent nitrogen.

51.5 g. of m-trifiuoromethylbenzonitrile dissolved in 50 cc. of ether isadded slowly to a solution of methyl-magnesium iodide prepared from 60g. of methyl iodide and 9 g. of magnesium in 400 cc. of ether. Thereaction mixture is stirred for about 3 hours and is poured into amixture of 500 g. of ice and 100 cc. of concentrated hydrochloric acid.The ether layer is separated from the'aqueous layer, dried withanhydrous magnesium sulfate and the ether evaporated. The residuecomprising m-trifluoromethylacetophenone is purified by distillation,m-Trifiuoromethylacetophenone thus prepared has a boiling point of about198-202 C. at atmospheric pressure. Analysis showed the presence of57.20 percent carbon and 3.82 percent hydrogen as compared with thecalculated values of 57.45 percent carbon and 3.75 percent hydrogen.

10 g. of m-trifluoromethylacetophenone, 2 g. of sulfur and 5.3 g. ofmorpholine are mixed and the mixture heated at about 135 C. for 16hours. 30 cc. of glacial acetic acid and 30 cc. of concentratedhydrochloric acid are then added to the reaction mixture and the mixturerefluxed for about '7 hours. The acetic acid is partially removed invacuo and the residue is poured into 500 cc, of water and the aqueousmixture extracted with three 300 cc. portions of ether. The etherextracts are combined, and further extracted with a solution of 10 g. ofsodium carbonate in 150 cc. of water. The alkaline extract is acidifiedwith hydrochloric acid whereupon m-trifiuoromethylphenylacetic acidprecipitates as an oil which crystallizes on standing. It is purified byrecrys-- tallization from petroleum ether. m-Trifiuoromethylphenylaceticacid thus prepared melted at about '72-'73" C. Analysis showed thepresence of 53.10 percent carbon and 3.38 percent hydrogen as comparedwith the calculated values of 52.95 percent carbon and 3.45 percenthydrogen.

19.5 g. of m-trifiuoromethylphenylacetic acid are dissolved in 300 cc.of methanol containing cc. of concentrated sulfuric acid and the mixtureis refluxed for about 5 hours. The bulk of the methanol is removed invacuo and the residue poured into cold water. The oily layer of methyltrifiuoromethylphenylacetate is separated from the aqueous layer andpurified by vacuum distillation. Methyl trifiuoromethylphenylacetatethus prepared boiled at about 103 C. at 12 mm. pressure. Analysis showedthe presence of 55.68 percent carbon and 4.26 percent hydrogen ascompared with the calculated values of 55.05 percent carbon and 4.16percent hydrogen.

8.2 g. of methyl m-trifluoromethylphenylacetate and 2.5 g. ofethanolamine are mixed and the mixture is heated to about 100 C. for 20hours. The heating is then continued for two hours longer during whichperiod the reaction mixture is subjected to vacuum to remove the excessethanolamine. Analysis of N-(Z-hydroxyethyl)-mtrifiuoromethylphenylacetamide thus prepared showed the presence of53.44 percent carbon and 5.28 percent hydrogen as compared with the cal-32 culated values of 53.44 percent carbon and 4.89 percent hydrogen.

Preparation of N-(2-hydro:cz/ethyl) -6-methoa:y-

Z-naphthylacetamide N (2'-hydroxyethy'l) -6-methoxy-2 -naphthylacetamiderepresented by the formula OHIO may be prepared in the following manner:

100 g. of 6-methoxy-2-acetonaphthone, 25.5 g. of sulfur and 87 g. ofmorpholine are heated at 135-140 C. for about 18 hours, and the excessmorpholine is then removed in vacuo. The residue is treated with 250 cc.of glacial acetic acid and 350 cc. of concentrated hydrochloric acid andthe mixture refluxed for about 24 hours. The mixture is then reduced toabout A of its volume by evaporation in vacuo and the residue treatedwith about 1 liter of water whereupon 6-methoxyz-naphthylacetic acidprecipitates, The G-methoxy-2-naphthylacetic acid is separated byfiltration and dissolved in a solution of 60 g. of sodium carbonate in500 cc. of water. The solution is treated with decolorizing carbon, isfiltered and the filtrate acidified with hydrochloric acid whereupon aprecipitate of 6-methoxy-2-naphthylacetic acid is obtained and separatedby filtration. The 6,-methoxy-2-naphthylacetic acid is dried anddissolved in about 1 liter of ether, and the solution is treated withdecolorizing carbon, filtered and the ether evaporated leaving the6-methoxy-2-naphthylacetic acid as a crystalline residue.6-methoxy-Z-naphthylacetic acid thus prepared melted at about 203-205 0.Analysis showed the presence of 71.74 percent carbon and 5.07 percenthydrogen as compared with the calculated values of 72.20 percent carbonand 5.60 percent hydrogen.

32.5 g. of 6-meth0xy-2-naphthylacetic acid are dissolved in 500 cc. ofmethanol containing 5 cc. of concentrated sulfuric acid. The mixture isrefiuxed for about two hours and then evaporated to a small volume. Theresidue is diluted with water and the methyl 6-methoxy-2-naphthylacetatewhich separates as an oil is removed by extraction with ether. The etherextract is washed with dilute sodium carbonate solution, dried overanhydrous magnesium sulfate and the ether evaporated. The residuecomprising methyl 6-methoxy-2-naphthylacetate is purified by vacuumdistillation. Methyl 6-methoxy-2-naphthylacetate thus prepared boiled atabout 192-193 C. at 1 mm. pressure and melted at about 86 C. Analysisshowed the presence of 72.72 percent carbon and 6.12 percent hydrogen ascompared with the calculated values of 73.03 percent carbon and 6.13percent hydrogen. 7

N-(2'-hydroxyethyl) -6-methoxy Z-naphthylacetamide is prepared byreacting 11.5 g. of methyl 6-methoxy-2-naphthylacetate and 3.5 g. ofethanolamine according to the method used for the preparation ofN-(Z-hydroxyethyl) -p-iodophenylacetamide.

N-(Z-hydroxyethyl) -6-methoxy Z-naphthylacetamide thus prepared meltedat about C. Analysis showed the presence of 68.95 percent carbon and6.85 percent hydrogen as compared with the calculated values of 69.04percent carbon and 6.61 percent hydrogen.

,auacee 3 Preparation of N-(2'-hydrozuethyl) -6-fluoro-2-naphthulacetamide N- (2'-hydroxyethyl) -6-fluoro-2-naphthylacetamiderepresented by the formula CHg-NH-CHaCHaOH may be prepared as follows:

78 g. of 2-methyl-6aminonaphthalene hydrochloride are mixed with 80 cc.of concentrated hydrochloric acid and 200 cc. of water. The mixture iscooled to about 5 C. and treated with stirring with a solution of 35 g.of sodium nitrite dissolved in 50 cc. of water. The resulting mixture ismaintained at a temperature of about 5 C. for one half hour and there isthen added thereto and with stirring about 130 g. of ice-cold 42 percentfluoroboric acid whereupon a precipitate of2-methyl-G-naphthalene-diazonium fluoroborate is formed. The precipitateis removed by filtration and dried in a vacuum desiccator over sulfuricacid. The dried 2-methyl-6-naphthalene-diazom'um fiuoroborate is placedin a distilling flask and heated locally with a small flame until avigorous exothermic reaction results. After subsidence of the vigorousreaction, the reaction mixture is distilled in vacuum whereuponsubstantially pure 2-methyl-6-fluoronaphthalene is obtained.2-methyl-6-fluoronaphthalene thus obtained melted at about 77 C.Analysis showed the presence of 82.53 percent carbon and 5.63 percenthydrogen as compared with the calculated values of 82.48 percent carbonand 5.66 percent hydrogen.

40 g. of 2-methyl-6-fluoronaphthalene are heated to about 210 C., andduring illumination with a 100 watt lamp, 40 g. of bromine are addedover a period of about 15 minutes. The reaction mixture comprising2-bromomethyl-6-fluoronaphthalene is purified by distillation in vacuo.2-bromomethyl-6-fiuoronaphthalene thus prepared boiled at 125-l30 C. at2 mm. pressure and melted at about 53 C. Analysis showed the presence of56.63 percent carbon and 3.15 percent hydrogen as compared with thecalculated values of 55.26 percent carbon and 3.37 percent hydrogen.

48 g. of 2-bromomethyl-6-fluoronaphthalene are added slowly to a hotsolution of 30 g. of potassium cyanide dissolved in a mixture of 60 cc.of water and 200 cc. of ethanol. The reaction mixture is refluxed forabout four hours, 40 g. of potassium hydroxide are then added andrefluxing is continued for five hours. The bulk of the alcohol isremoved by evaporation in vacuo and 300 cc. of water are added to theresidue. The resulting solution is treated with decolorizing carbon andfiltered, and the filtrate acidified with hydrochloric acid whereupon6-fiuoro-2-naphthylacetic acid separates in crystalline form. 6-fiuoro-2-naphthylacetic acid thus prepared melted at about 138-139 C.Analysis showed the presence of 70.68 percent carbon and 4.60 percenthydrogen as compared with the calculated values of 70.58 percent carbonand 4.44 percent hydrogen.

30 g. of 6-fiuoro-2-naphthylacetic acid are dissolved in 300 cc. ofmethanol containing 10 cc. of concentrated sulfuric acid and the mixtureis refluxed for about hours. The bulk of the alcohol is then distilledfrom the reaction mixture and the residue diluted with cold waterwhereupon methyl 6-fiuoro-2-naphthylacetate separates aezan oily layer.The methyl ester is extracted with ether, the ether solution dried overanhydrous magnesium sulfate and the ether distilled. The residuecomprising methyl 6 fluoro-2-naphthylacetate is purified by vacuumdistillation. Methyl 6-fluoro-2 naphthylacetate thus prepared boiled at163-166 C. at 2 mm. pressure and melted at about 48-49 C. Analysisshowed the presence of 71.35 percent carbon and 5.25 percent hydrogen ascompared with the calculated values of 71.55 percent carbon and 5.08percent hydrogen.

11 g. of methyl 6-fluoro-2-naphthylacetate and 3.5 g. of ethanolamineare reacted according to the procedure described in the preparation ofN- (2-hydroxyethyl) -p-iodophenylacetamide.

N-(2'-hydroxyethyl) -6 fluoro 2 naphthylacetamide thus prepared meltedat about -146 C. Analysis showed the presence of 67.88 percent carbonand 5.60 percent hydrogen as compared with the calculated values of68.00 percent carbon and 5.71 percent hydrogen.

Preparation of N-(z hydroryethyl)-6-bromo- Z-naphthylacetamide N (2'hydroxeythyl) 6-bromo-2-naphthylacetamide represented by the formula 0ORAL-NH-CHaCBhOH may be prepared in the following manner:

63 g. of 2-methyl-fi-aminonaphthalene are added to a mixture of 700 g.of 48 percent hydrobromic acid and 100 cc. of water. The mixture is0001811170 below 5 C. and during stirring a solution of 50 g. of sodiumnitrite dissolved in 100 cc. of water is added over a period of aboutfour hours. The reaction mixture is poured slowly into a hot solution ofg. of cuprous bromide dissolved in 800 cc. of 48 percent hydrobromicacid. The mixture is allowed to stand at room temperature for about 12hours and is then steam distilled whereupon 2-methyl-6 bromonaphthaleneis obtained in the distillate wherefrom it is separated by filtration.2-methyl-6-bromonaphthalene thus prepared melted at about 142 C.Analysis showed the presence of 59.75 percent carbon and 4.07 percenthydrogen as compared with the calculated values of 59.75 percent carbonand 4.10 percent hydrogen.

2-methyl-6-bromonaphthalene is converted to 2-bromomethyl 6bromonaphthalene according to the procedure described for thepreparation of 2-bromomethyl-6-fiuoronaphthalene. 2-bromomethyl 6bromonaphthalene thus prepared melted at about 124-125 C. Analysisshowed the presence of 44.08 percent carbon and 2.64 percent hydrogen ascompared with the calculated values of 44.04 percent carbon and 2.69percent hydrogen.

2-bromomethyl-6-bromonaphthalene is converted into6-bromo-2-naphthylacetic acid according to the procedure described forthe preparation of 6 fluoro 2 naphthylacetic acid.6-bromo-2-naphthylacetic acid thus prepared melted at about -176 C.Analysis showed the presence of 54.45 percent carbon and 3.30 percenthydrogen as compared with the calculated values of 54.36 percent carbonand 3.42 percent hydrogen.

Methyl 6-bromo-2-naphthylacetate is prepared according to the proceduredescribed in the preparation of methyl 6-fiuoro-2-naphthylacetate.Methyl 6-bromo-2-naphthylacetate thus prepared melted at about 67-69 C.Analysis showed the presence of 55.36 percent carbon and 3.61 percenthydrogen as compared with the calculated values or 55.93 percent carbonand 3.97 percent hyd en.

Methyl 6-bromo-2-naphthylacetate is converted to N'- (2'-hydroxyethyl)-6-bromo-2-nap hthylacetamide according to the procedure described inthe preparation of N (2-hydroxyethyl) p-iodophenylacetamide. N (2'hydroxyethyD- 6 bromo 2 naphthylacetamide thus prepared melted atabout'167-168" C. Analysis showed the presence of 54.55 percent carbonand 4.58 percent hydrogen as compared with the calculated values of54.56 percent carbon and 4.58percent hydrogen.

Preparation of N- (Z-hydrozyethvl) -'mtrifluoromethylphenocyacetamide N(2 hydroxyethyl) -m-trifiuoromethylphenoxyacetamide represented by theformula I CF;

o-cm -NH-cmcmorr may be prepared in the following manner;

19 g. or m-trifiuoromethylphenol and .19 cc.

of 12.5 N sodium hydroxide solution are treated with 11 g. ofchloroacetic acid. The mixture is heated at about 80-100" 0. for aboutthree hours. The reaction mixture is then diluted with 400 cc. of water,acidified with hydrochloric acid and the m-trifiuoromethylphenoxyaceticacid which.

separates isextracted with ether. The ether extract is in turn extractedwith'a dilute aqueous solution of sodium bicarbonate. The sodiumbicarbonate solution is acidified whereuponm-trifiuoromethylphenoxyacetic acid is precipitated in solid form.m-Trifluoromethylphenoxyacetic acid thus prepared melted at about 92-930.

Analysis showed the presence of 49.59 percent carbon and 3.26 percenthydrogen as compared with the calculated values of 49.10 percent carbonand 3.21 percent hydrogen.

m-Trifiuoromethylphenoxyacetic acid is converted to methylm-trifluoromethylphenoxyacetate according to the procedure described inthe preparation of methyl 6-fluoro-2-naphthylacetate. Methylm-triiluoromethylphenoxyacetate thus prepared boiled at about 101 C. at3 mm. pressure. Analysis showed the presence of 51.01 percent carbon and3.68 percent hydrogen as compared with the calculated values of 51.29percent carbon and 3.87 percent hydrogen.

16.6 g. of methyl m-trifluoromethylphenoxyacetate and 5 g. ofethanolamine are treated according to the procedure described in thepreparation Of N-(2-hydroxyethyl) -p-iodophenylacetamide. N-(2-hydroxyethyl) -m-trifluoromethylphenoxyacetamide thus obtained meltedat about 86 C. Analysis showed the presence of 5.44 percent nitrogen ascompared with the calculated value of 5.32 percent nitrogen.

Preparation of N-(2-hudroa:uethyl) -p-methylphenoayacetamideN-(2-hydroxyethyl) p methylphenoxyacetamide represented by the formula 0era-@ocmE-nn-cmcmon may be prepared in the following manner:

Methyl p-methylphenoxyacetate is prepared from p-methylphenoxyaceticacid according to the procedure described for the preparation of methyl6-fluoro-2-naphthylacetate. Methyl pmethylphenoxyacetate thus preparedboiled at about 119 C. at 5mm. presure. Analysis showed 7 the presenceof 66.56 percent carbon and 6.85

percent nitrogen.

' Preparation of N-.(2'-hydr0:cyethz/l) -2-cyclopentene-I-acetamide 1 N-(2'-hydroxyethyl) -2-cyclopentene 1 acetamide represented by the formulamay be prepared in the following manner:

15 g. of diethyl 2-cyclopentenylmalonate, 15 g. of potassium hydroxideand 15 cc. of water are mixed and heated at about C. for five hours. Thereaction mixture is cooled and acidified with concentrated hydrochloricacid and the mixture extracted several times with ether. Evaporation ofthe ether leaves a residue of 2-cyclopentenylmalonic acid which isheated to about 160 C. whereupon decarboxylationoccurs and2-cyclopentene-l-acetic acid is formed. The 2-cyclopentene-l-acetic acidis dissolved in ether and treated with an ethereal solution ofdiazomethane The ethereal solution is then shaken with dilute sodiumbicarbonate solution, the ether solution separated and dried overanhydrous magnesium sulfate. Evaporation of the ether leaves a residueof methyl 2-cyclopentene-1-acetate.

10 g. of methyl Z-cycIopentene-l-acetate and 4.5 g. of ethanolamine areheated together at about C. for four hours. The excess ethanolamine isremoved from the reaction mixture by heating the mixture to about 150 C.in a vacuum for about three hours. The residue comprising N(2-hydroxyethyl) 2 cyclopentene 1 acetamide may be purified bydissolving it in ethyl acetate and reprecipitating it by the addition ofpetroleum ether. N-(2'-hydroxyethyl) -2-cyc1opentene-l-acetamide thusprepared was obtained in the form of an oil. Analysis showed thepresence of 8.7 percent nitrogen as compared with the calculated valueof 8.3 percent nitrogen.

Preparation of N-allyl-styrylacetamide N-allyl-styrylacetamiderepresented by the formula may be prepared by reacting 18 g. ofstyrylacetyl chloride and 11.4 g. of allylamine according to theprocedure described in the preparation of N 'y- (pbromophenyl)-butyrylvaline.

N-allyl-styrylacetamide thus prepared melted at about 61.5-63 C.Analysis showed the presence of 77.83 percent carbon, 7.58 percenthydrogen and 6.76 percent nitrogen as compared with the calculatedvalues or 77.58 percent carbon, 7.51. per cent hydrogen and 6.96 percentnitrogen.

Preparation of N-p-carbethozyhydroxuphenulacetylvaline N pcarbethoxyhydroxyphenylacetylvaline represented by the formula 0 (H510 OC OQCHaE-NH-ifl about 78-79 C. Analysis showed the presence of 58.90percent carbon and 5.37 percent hydrogen as compared with the calculatedvalues of 58.92 percent carbon and 5.32 percent hydrogen.

11.2 g. of p-carbethoxyhydroxyphenylacetic acid and 10.4 g. ofphosphorus pentachloride are mixed whereupon a vigorous reactionresults. The reaction mixtureis allowed to stand at room temperature forabout 15 hours and is then subiected to a vacuum to remove volatilematerial. The residue comprising p-carbethoxyhydroxyphenylacetylchloride is dissolved in 50 cc. o1 ether and is added simultaneouslywith 50 cc. of 1 N sodium hydroxide solution to a solution of 6.43 g. ofvaline dissolved in 55 cc. of 1 N sodium hydroxide solution. Thereaction mixture is stirred for one hour, the ether layer is separatedand discarded and the aqueous layer is acidified to a pH of about 4whereupon a precipitate of N-p-carbethoxyhydroxyphenylacetylvalineseparates. The precipitate is separated by filtration and recrystallizedfrom a mixture of ethanol and ether. N pcarbethoxyhydroxyphenylacetylvaline thus prepared melted at about125-127 C. Analysis showed the presence of 4.50 percent nitrogen ascompared with the calculated value of 4.33 percent nitrogen.

Preparation of N-(z-hydrozuethyl) -pnaphthylacetamide N-(2 hydroxyethyl)p naphthylacetamlde represented by the formula 0 CHaE-NH-CHaCHaOH may beprepared by reacting 6.4 g. of ethyl flnaphthylacetate and 1.9 g. ofethanolamine ac- I Preparation of N-o-chlorophenulacetulvalineN-o-chlorophenylacetylvaline represented by the formula on; CH 0 \Q Qcm-mr-tn 1 door! may be prepared by converting 8.5 g. ofo-chlorophenylacetic acid to the corresponding acid chloride andreacting the latter with valine according to the procedure described inthe preparation of N-p-chlorophenylacetylvaline.N-ochlorophenylacetylvaline thus prepared melted at about 122124 C.Analysis showed the presence of 4.80 percent nitrogen as compared withthe calculated value of 5.19 percent nitrogen.

Preparation of N-(Z-hydroxyethyl) -mchlorophenylacetamideN-(2-hydroxyethyl) m chlorophenylacetamide represented by the formula oQ-omtt-mr-omomon may be prepared by-reacting 58 g. of ethylmchlorophenylacetate and 19 8'. of ethanolamine according to theprocedure described in the preparation ofN-(2-hydroxyethyl)-o-fluorophenylacetamide.

N-(2 hydroxyethyl) m chlorophenylacet amide thus prepared melted atabout 114-117" C. Analysis showed the presence of 6.54 percent nitrogenas compared with the calculated value of 6.55 percent nitrogen.

Preparation of N-(Z-hydroxyethyl) -mmethylphenylacetamide N-(2 Qhydroxyethyl) m methylphenylacetamide represented by the formula oQ-omtB-mr-cmcmon may be prepared by heating 8.9 g. of ethylmmethylphenylacetate and 4 g. of ethanolamine 50 at 135-140 C. for about12 hours. The reaction mixture is then heated to about 80 C. andsubjected to a vacuum of about 0.01 mm. for about 10 hours to removevolatile constituents.

N (2-hydroxyethyl) m-methylphenylaceta- 55 mide thus prepared was anoil. Analysis showed the presence of 7.21 percent nitrogen as comparedwith the calculated value of 7.25 percent nitrogen.

, Preparation of N-(Z-hydroxuethyl) -p-:renulacetamideN-(2-hydroxyethyl)-p-xenylacetamide represented by the formula 0 w@Qom-mr-cmomon may be prepared as follows:

7 g. of p-xenylacetic acid are dissolved in 150 cc. of absolute ethanolcontaining 1 cc. of concentrated sulfuric acid. The solution is refluxedfor about 12 hours and the bulk of the alcohol is evaporated in vacuo.The residue comprising ethyl p-xenylacetate is treated with dilutesodium hydroxide solution to neutralize the sulfuric acid present andthe ethyl p-xenylacetate which separates from the aqueous layer as anoil is removed.

The crude ethyl p-xenylacetate is reacted with 2.4 g. of ethanolamineaccording to the procedure described in the preparation ofN-(2-hydroxyethyl) -o-fluorophenylacetamide. N-(2-hydroxyethyl)-p-xenylacetamide thus prepared melted at about 172-175 C. Analysisshowed the presence of 5.70 percent nitrogen as compared with thecalculated value of 5.75 percent nitrogen.

Preparation of N-m-nitrophenylacetylvaline N-m-nitrophenylacetylvalinerepresented by the formula phenylacetyl chloride is reacted with 6.0 g.of w valine according to the procedure used in the preparation ofN-y-(p-bromophenyl)-butyry1- valine. N-m-nitrophenylacetylvaline thusprepared melted at about 153-158 C. Analysis showed the presence of10.10 percent nitrogen as compared with the calculated value of 10.00percent nitrogen.

Preparation of N- (Z-hydroryethyl) -pmethylmercaptophenylacetamideN-(Z-hydroxyethyl) p-methylmercaptophenylacetamide represented by theformula 0 ll H3O scnc-Nn-omomon may be prepared in the following manner:

24.8 g. of thionaisole, 24 g. of acetyl chloride and 150 cc. of carbonbisulfide are cooled to about 0 C. and treated with small portions ofanhydrous aluminum chloride until a total of g. are used. The reactionmixture is stirred four hours and is then poured into a mixture of iceand hydrochloric acid. The carbon bisulfide is distilled from thereaction mixture and the aqueous residue is extracted with ether. Theether extract is dried over magnesium sulfate and evaporated leaving asolid residue of p-methylmercaptoacetophenone which is purified byrecrystallization from a mixture of ether and petroleum ether.p-Methylmercaptoacetophenone thus prepared melted at about '72-'75showed the presence of 65.06 percent carbon and 5.96 percent hydrogen ascompared with the calculated values of 65.02 percent carbon and 6.06percent hydrogen.

49.8 g. of p-methylmercaptoacetophenone are converted top-methylmercaptophenylacetic acid according to the procedure describedfor the conversion of 2-acetotetralin to 5,6,7,8-tetrahydro-2-naphthylacetic acid. p-Methylmercaptophenylacetic acid thus preparedmelted at about 92-94 0. Analysis showed the presence of 59.71 percentcarbon and 5.25 percent hydrogen as compared with the calculated valuesof 59.31 percent carbon and 5.53 percent hydrogen.

p-Methylmercaptophenylacetic acid is converted to the correspondingmethyl ester by refluxing a solution of p-methylmercaptophenylaceticacid in methanol saturated with dry hydrogen chloride gas. The bulk ofthe alcohol is distilled from the reaction mixture and the residuepoured into cold water. The oily layer of methylp-methylmercaptophenylacetate which forms is separated and purified bydistillation. Methyl p-methylmercaptophenylacetate thus prepared boiledat about 179 181 C. at 3 mm. pressure. Analysis showed the-presence of60.76 percent carbon and 6.13 percent hydrogen as compared with thecalculated values of 61.19 percent carbon and 6.16 percent hydrogen.

18 g. of methyl p-methylmercaptophenylacetate and 6.5 g. of ethanolamineare reacted according to the procedure described in the preparation of N(2 hydroxyethyl) -o-fluoropheny1acetamide. N (2-hydroxyethyl)-p-methylmercaptopheny1- acetamide thus prepared melted at about 115-117C. Analysis showed the presence of 6.30 percent nitrogen as comparedwith the calculated value of 6.30 percent nitrogen.

Preparation of N-(Z-hydroxyethyl) -m-bromophenylacetamideN-(2-hydroxyethyl) m bromophenylacetamide represented by the formula maybe prepared by reacting 37 g. of ethyl mbromophenylacetate and 11 g. ofethanolamine according to the procedure described in the preparation ofN-(Z-hydroxyethyl)-o-fluorophenylacetamide.

N- (Z-hydroxyethyl) -m-bromophenylacetamide thus prepared melted atabout 129-130 C. Analysis showed the presence of 5.37 percent nitrogenas compared with the calculated value of 5.43 percent nitrogen.

Preparation of N-(2'-hydro:cyethyl) -2,3-dimethomyphenylacetamide N (2'hydroxyethyl) -2,3 dimethoxyphenyl- C. Analysis 00 added a solution of75 g.

represented by the formula Preparation of N-(Z-hydroxyethyl)p-phenomyphenylacetamide N (2 hydroxyethyl) p phenoxyphenylacetamiderepresented by the formula 0 ll .QOQCHgC-NH-CHICILOH may be prepared inthe following manner:

60 g. of p-phenoxyacetophenone, 13 g. of sulfur and 40 fiuxed overnight.To the reaction mixture is of potassium hydroxide acetamide g. ofmorpholine are mixed and re-

